IgE.mediated allergic disease appears to be very common particularly in industrialized countries where up to one quarter of the population is affected by allergic rhinitis. (Settipane, R. A., Allergy Asthma Proc, 22(4):185-9 (2001)). Furthermore people suffering from allergic rhinitis show a lower quality of life than healthy one, (Bousquet, J., et al., J Allergy Clin Immunol, 94(2):182-8 (1994)) with only a few going into remission spontaneously.
Ragweed (Ambrosia artemisiifolia) belongs to the Asteraceae plant family and represents the most important allergenic source within this genus. The Ambrosia sp. are native to eastern and central North America, but are also persistently spreading in Central and Southern Europe. The population allergic to ragweed pollen is increasing particularly in the US (Gadermaier, Allergy, 63(11): 1543-9 (2008)). In northern America, 45% of allergic individuals suffer from ragweed pollen allergy. In Europe, atopic sensitization in ragweed-populated areas (e.g. France, Italy, Austria, Hungary, Croatia and Bulgaria) is constantly increasing.
A few allergen proteins have been associated with ragweed allergy. Sequences of such ragweed allergens (Amb a) can be found in the Nucleotide protein databases (NCBI). The allergens can be allocated to 6 groups according to their sequences (NCBI database) (Wopfner et al, IAAI, 138(4):337-46 (2005)), namely:                Amb a 1/Amb a 2 (95% of patients), pectate lyase family        Amb a 3/Amb a 7 (30-50%; 10-20% respectively), plastocyanins basic prots        Amb a 5 homologues        Amb a 6 (21% of patient), lipid transfer protein        Amb a 8 (35% of patients) profilin        Amb a 9 (10-15% of patients) Ca-binding protein        
It is widely accepted that the Amb a 1/a 2 group clearly dominates in the population allergic to ragweed. In addition, four variants of Amb a 1 have been described, namely 1.1, 1.2, 1.3 and 1.4 which share high sequence identity. A small proportion of the IgE of the patients exclusively reacted with recombinant Amb a 1.1, whereas the IgE of most patients reacted with Amb a 1.1 as well as Amb a 1.2 and Amb a 1.3 proteins. Another family member of Amb a 1 termed Amb a 1.4 was identified, which appears to be only a minor component of the Amb a 1 family. In addition, Amb a 2 shares 65% identity with Amb a 1 (Wopfner et al, 2005). Since a large majority of the Amb a 1 positive patients react to Amb a 1.1, COPs from that sequence were selected.
Amb a 1 is an acidic (pI 4-6), non-glycosylated single-chain protein with a molecular weight of approximately 38 kDa. It may undergo proteolysis during chromatographic purification and was found to be cleaved into an alpha chain of 26 kDa non-covalently associated with a beta chain of 12 kDa (Wopfner et al, Mol Immunol 46(10):2090-7 (2009)).
The only treatment directed to the cause of IgE-mediated allergy is specific immunotherapy (SIT). The treatment consists in injecting increasing doses of allergens for extended periods of time (three to five years) to induce tolerance in the allergic patient. Several studies showed the benefit of this therapy on the allergic response, in particular upon long-term treatment. (Drachenberg, K. J. et al., Allergol Immunopathol, 31(2):77-82 (2003) and Dam Petersen, K. et al., Allergol Immunopathol 33(5)264-269 (2005)). However, a number of side effects were observed particularly during ultra rush therapies, where up to 30% of the patients have to be treated for allergic symptoms during the course of therapy. (Birnbaum et al., Clin. Exp. Allergy, 33(1):58-64 (2003)). There is thus a strong medical need for an alternative to conventional SIT in the form of a shorter treatment with acceptable safety.
Different approaches have been tested to improve the safety and efficacy of SIT. Formulations or existing extracts have been improved by adding adjuvants, like MPL (Allergy Therapeutics), (Drachenberg, K. J. et al., (2003)) DNA sequences (Hartl, A. et al., Allergy, 59(1):65-73 (2004)) or bacteriophage combined with CpG (Martinez Gomez, J. M. et al., Pharm. Res., 24(10):1927-35 (2007)) which increase the TH1 immune response, thus allowing possible reductions in the amount of allergen extract. Defined allergens were used instead of whole extracts. In the case of birch pollen, a clinical trial with recombinant Bet v 1 has shown efficacy equivalent to whole birch pollen extract (Pauli, G. et al., J. Allergy Clin. Immunol, 122(5):951-60 (2008)). In the case of ragweed, Amb a 1 associated with immunostimulatory DNA sequences appeared to offer long term clinical efficacy on a pilot study (Creticos et al., N Engl J Med, 355:1445-55 (2006)).
To diminish the occurrence of allergic symptoms resulting from treatment, different groups explored the use of products with hypoallergenic potential, namely showing reduced IgE binding. In particular, peptides encompassing a restricted number of T-cell epitopes were used for allergen immunotherapy of cat dander with limited efficacy (Campbell, J D et al., J Exp Med., 206(7):1535-47 (2009)). However, allergens harbor a great variety of T cell epitopes partly dependent on the HLA type of the patient. For example, T cell epitopes were found scattered throughout the Amb a 1 sequence (Jahn-Schmid B. et al., J Allergy Clin Immunol, 126(5):1068-1071 (2010)). Thus an efficient immunotherapy product should preferably contain the complete sequence of the allergen rather than selected T-cell epitopes.
The use of fragments of allergens remains attractive, based on the evidence that human IgE recognize mainly non-contiguous epitopes which may be separated by fragmentation of the allergen. Two contiguous fragments of Bet v 1 or trimeric forms of Bet v 1 were tested in a phase I study in human and showed a trend towards improvement of well being but provided no significant improvement in symptom medication scores (Niederberger, V. et al., Proc Natl Acad Sci USA, 101(2):14677-82 (2004)). In that study, however, a number of adverse events were observed, the majority of which occurred hours after the injections (Purohit, A. et al, Clin Exp Allergy (2008)). Three fragments of the major allergen of bee venom, namely phospholipase A2, were also tested in humans, showing an excellent safety due to lowered IgE binding while eliciting elevated levels of IgG4 and IL-10 (Fellrath et al., J. Allergy Clin. Immunol, 111:854-861 (2003)). A method was devised to select contiguous overlapping peptides (COPs) for treatment of allergy which together form the entire amino acid sequence of an allergen, thus providing all possible T cell epitopes of the allergen, while having lowered IgE binding (U.S. Pat. No. 7,923,209). Such selected fragments show a reduced ability to reform the original tertiary structure of the allergen, if any, resulting in a reduced ability to bind IgE and therefore to elicit allergic reactions in humans.